This research program will investigate molecular mechanisms by which insulin regulates cellular metabolism. Reversible phosphorylation of several key enzymes operates to control their activity, thereby governing the synthesis and degradation of glycogen, fats and cholesterol. Insulin promotes the net dephosphorylation of the principle biosynthetic enzymes of these pathways through activation of phosphoprotein phosphatase. Unfortunately, little is known about the phosphatases and attempts to purify and characterize them have been plagued by several problems. Most native phosphatases are inactive and their subunit structures and mechanisms of regulation remain to be explored. From my own investigations it appears that a trypsin-resistant Mr = 35,000 phosphatase, postulated as a common catalytic subunit of larger holoenzymes is only the catalytic domain of previously undetected, inactive subunits that yield the active species upon limited proteolysis. This research will employ antibodies against the catalytic domain in a "Western" electroblotting procedure with peroxidase-immunoassay to identify active and inactive proteins that are structurally related. Peptide mapping by high performance liquid chromatography will be used to determine whether the different size phosphatases are encoded in a single gene or in multiple structural genes. The subunit structure of the native, inactive holoenzymes will be revealed by combining traditional purification methods with the electroblot analysis. This strategy should allow detection of stable changes in phosphatases produced by insulin treatment of isolated cells. The results should provide new, basic information on the structure and regulation of the phosphatases and on the molecular mechanisms of insulin action.